Fungicides are compounds of natural or synthetic origin that protect plants against damage caused by fungi. Current methods of agriculture rely heavily on the use of fungicides. In fact, some crops cannot be grown usefully without the use of fungicides. Using fungicides allows a grower to increase the yield of the crop and consequently, increase the value of the crop. Many synthetic fungicides are classified as carcinogens by the Environmental Protection Agency (EPA) and are toxic to wildlife and other non-target species. In addition, chemical fungicides are harmful towards vertebrates (humans), persist in the soil environment, and can contaminate ground water supply. Furthermore, prolong chemical fungicide application results in target-surviving fungi developing an evolutionary resistance to the chemical fungicide. In order to eradicate chemical resistant-fungi, a cycle of even more potent chemical fungicides are utilized, resulting in more environmental damage and eventually even more chemical resistant fungi.
Among pecans, peaches, and other fruit and nut trees, fungal and oomycete incited diseases are a significant concern for commercial productivity. In Southeastern parts of the United States, pathogens including, but not limited to Glomerella cingulata, Phomopsis spp., Phytophthora cactorum, and Fusicladosporium effusum have a substantial impact on pecan production. Phytophthora species are homothallic oomycetes which produce oospores that spread by rain splash or irrigation water. Glomerella, Phomopsis, and Fusicladosporium species produce conidia or ascospores that spread by rain splash or wind. The fungus Monilinia fructicola also has a substantial impact on peach production.
Fungal pathogens such as Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, Fusicladosporium effusum, and Monilinia fructicola are controlled by chemical fungicides such as dodine, fenbuconazole, and triphenyltin hydroxide. However, environmental concerns, toxicological effects, and concern over target-organism resistance warrants development of alternative control methods. Methods for biocontrol via bacteria biotoxins have been recognized. For example, see Smart, G. C. (1995) “Entomopathogenic Nematodes for the Biological Control of Insects”, Journal of Nematology (Supplement) 27(4S):529-534, U.S. Pat. Nos. 6,048,838, and 5,549,889.
Insect pathogenic nematodes of the families Steinernematidae and Heterorhabditidae are symbiotically associated with bacteria of the genera Xenorhabdus and Photorhabdus respectively. The entomopathogenic nematodes generally form enduring juveniles that are adapted for long-term survival in soil conditions. The symbiotic bacteria are released into the haemolymph after penetration of the juvenile into a suitable insect host. The nematodes provide shelter to the bacteria, which, in return, kill the insect host and provide nutrients to the nematode. Symbiotic bacterial cannot survive in nature without the nematode, but can survive in sterile in vitro culture without the nematode host. Through various extraction methods, it has been observed that these nematode bacteria have the ability to kill a wide range of different insects without the aid of their nematode partners. While nematodes are used commercially on a wide range of insects, it is the endogenous symbiotic bacteria that are central to nematode virulence. The bacterial produced toxins and antibiotics are lethal towards insects, microbes and a variety of fungi.
Bacterial toxins, such as antibiotics, have been used to control pathogens. The toxin can be isolated and applied directly to the plant or the bacterial species may be administered so it produces the toxin in situ. It has been long known that bacteria and bacterial metabolites that have antimicrobial properties have been investigated for suppression of insect population, as elaborated by Smart, G. C. (1995) “Entomopathogenic Nematodes for the Biological Control of Insects”, Journal of Nematology (Supplement) 27(4S):529-534.
Xenorhabdus spp. are symbionts of entomopathogenic Steinernema spp. nematodes, while Photorhabdus spp. are associated with Heterorhabditis spp. In nature, the bacteria exists in the intestine of their nematode symbionts or in the insect hosts that nematodes infect; the bacteria require the protection of the nematode to survive in the external environment. Given such bacteria are toxic to insects, it is well known in the art that bacterial produced toxins can be utilized as insecticides. For instance, U.S. Pat. No. 6,048,838 discloses a protein toxin isolated from Xenorhabdus strains as an insecticides. It is known in the art that Xenorhabdus spp. and Photorhabdus spp. metabolites with antibiotic activity can be cultured in vitro on solid media or in liquid fermentation. For instance see Paul V. J. et al., (1980) “Antibiotics in microbial ecology” Journal of Chemical Ecology 7:589-597, Barbercheck M. E. et al., (1996) “Effect of Cucurbitacin D on in Vitro Growth of Xenorhabdus and Photorhabdus spp., Symbiotic Bacteria of Entomopathogenic Nematodes” Journal of Invertebrate Pathology 68(2):141-145, U.S. Pat. No. 6,316,476, and WO 95/03695.
Chemical secondary metabolites from symbiotic bacteria have been identified. Specifically chemical groups of Xenorhabdins, Xenorxides, Xenocoumacins, Indoles, Nematophics, Hydroxystilbenes, and Puromycins derived from Xenorhabdus and Photohabdus. Indeed, some antimicrobial compositions, such as xenorxides and nematophin, have been identified as outlined in U.S. Pat. Nos. 6,316,476 and 5,569,668 respectively.
Although Xenorhabdus spp. and Photorhabdus spp. and their metabolites have been tested for suppression of some fungal or oomycete species pathogens such as, Phytophthora infestans, no tests have determined the potency of these agents against specific phytopathogens of pecan and peach plants. Fungicidal properties associated with these symbionts are known to vary among bacterial species and strain. Additionally, potency of bacterial metabolites from P. luminescens (VS), Photorhabdus sp. (MX4), and Xenorhabdus sp. (3-8b), have not been assessed for antibiotic activity against any organism, and the nematodes/bacteria complexes from which these metabolites were derived remain relatively unstudied. Thus, there is a need in the art to determine whether metabolites from Xenorhabdus and Photorhabdus species would be effective fungicide to protect peach and pecan crop from fungi such as Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, Fusicladosporium effusum, and Monilinia fructicola. 
In addition, there is a need to determine whether the bacterial metabolites of Xenorhabdus and Photorhabdus genera has any phytotoxicity throughout in vivo experimentation to determine whether any fungal suppressive effects are limited to only in vitro conditions.